The impact of C. elegans ceramide glucosyltransferase enzymes on the beneficial effects of B. subtilis lifespan

Ceramide glucosyltransferase (CGT) adds sugar moieties to ceramide, forming glucosylceramides that play roles in immune signaling, stress response, and host-bacterial interactions. Here, we examined whether mutations in cgt block the beneficial effects of Bacillus subtilis on C. elegans lifespan. We found that loss of cgt-1 or cgt-3 reduces lifespan compared to wildtype worms, but did not prevent the lifespan-extending phenotype of B. subtilis . However, cgt-1(ok1045) and cgt-3(tm504) did play a minor role in blocking stress resistance of 5-day old worms treated with B. subtilis . Further studying CGTs may elucidate potential roles of glucosylceramides in host-bacterial interaction.


Figure 1. Loss of CGT does not impair the beneficial effects of B. subtilis:
A) Survival curves of wild-type (N2) and cgt mutants (cgt-1(tm1027), cgt-1(ok1045), cgt-2(tm1192), cgt-3(tm504)) fed on E. coli (OP50; yellow) or B. subtilis (3A1T; blue). Worms were tracked for lifespan starting at L4 stage (day 0) and scored every 2 days. Bagged or missing worms were censored (indicated by a crossline). Significant differences were found between survival of animals treated with E. coli vs B. subtilis. The bottom right panel in (A) shows the survival curves of all animals fed E. coli (OP50) on the same graph. B) Table showing sample size, mean, standard error deviation, & median values for the lifespans of worms grown on the control bacteria (E. coli) and experimental bacteria (B. subtilis). For B, * indicates significant difference compared to the respective animal on B. subtilis and # indicates significant difference compared to N2 on E. coli. C) Acute stress response survival curves of wild-type (N2) and cgt mutants treated with 100mM paraquat. Experiments were performed on 1-day, 5-day, and 10-day old animals. Worms were grown to respective ages on either E.coli (OP50; yellow) or B. subtilis (3A1T; blue) bacterial lawns prior to the stress test. For all, survival curves were analyzed using Kaplan-Meier estimates, and pairwise comparisons were performed using a log-rank test.

Description
At the membrane surface of intestinal cells, there is a rich complement of glucosylceramides. Ceramides are a type of sphingolipid that play a role in lipid microdomains, stress response, and cell death (Rohrhofer et al. 2021). The enzyme ceramide glucosyltransferase (CGT) catalyzes the addition of sugar moieties onto ceramide in the lipid bilayer. However, it is not known how bacteria-host interactions are affected by glucosylceramide metabolism. Could the presence of different cgt enzymes affect the impact of the beneficial effects of bacteria on the host physiology? There are three genes (cgt-1, cgt-2, and cgt-3) that are thought to have CGT enzymatic activity in C. elegans. Previous studies suggest that cgt-1 and cgt-3 have a greater number of amino acids relating to functional enzymatic activity; thus, mutations in cgt-1 and cgt-3 may have more of a negative impact on animal physiology than cgt-2 (Marza et al. 2009). Specifically, cgt-1;cgt-3 double mutants have larval phenotypes, cgt-1 and cgt-3 are highly expressed in the worm intestine, and they are known to serve developmental roles; furthermore, loss of all cgts (cgt-1, cgt-2, and cgt-3) are lethal (Marza et al. 2009). Re-expression of cgt enzymes in the intestine can rescue larval phenotypes of cgt-1;cgt-3 double mutants, suggesting CGTs have important intestinal functions (Marza et al. 2009). CGTs have also been shown to help establish intestinal cell polarity during development (Zhang et al. 2011). More recently, there has been observation of CGTs acting on autophagolysosomes to recruit clathrin and mediate lysosome recycling (Wang et al. 2021).
The commensal bacteria Bacillus subtilis has been demonstrated to increase lifespan and promote survival to the oxidative stressor juglone and thermotolerance of the nematode Caenorhabditis elegans (Donato et al. 2017;Smolentseva et al. 2017). Interestingly, these effects were dependent on the biofilm forming nature of B. subtilis. Biofilms are protective structures composed of extracellular matrix proteins and signaling molecules, providing a place for bacteria to grow and survive (Vlamakis et al. 2013). It also acts as a point of contact between bacteria and the host intestinal membrane. However, less is known about whether glucosylceramides mediate the beneficial effects of B. subtilis. Recent studies show that glycosylated ceramides are targets of Bacillus thuringiensis binding, leading to C. elegans infection (Griffitts et al. 2005). Furthermore, CGT inhibition can weaken the colon cell barrier to the Bacteroides fragilis toxin (Patterson et al. 2020).
Given the effect of B. subtilis on stress response and lifespan, along with the roles of cgt enzymes in the intestine, we aimed to examine whether the protective effects of B. subtilis require cgt enzymes (cgt-1, cgt-2, and cgt-3). To do this, we compared lifespan and stress response of wild-type and mutant animals when grown on either the B. subtilis wild-type isolate (3AIT) or the common lab bacteria E. coli (OP50). First, we found that B. subtilis increased the survival of wild-type animals approximately 10% ( Figure 1A), which is similar to the 15% increased survival demonstrated in other studies (Donato et al. 2017). However, B. subtilis also increased the survival of all cgt mutant animals examined. When comparing wild-type animals to cgt mutants on OP50, we found that mutations in cgt-1 and cgt-3 reduced lifespan compared to wild-type ( Figure  1A,B). Similarly, Wang et al. (2021) found that cgt-3 RNAi reduced lifespan. Whereas loss of particular ceramide glucosyltransferase genes can reduce lifespan, it does not block the beneficial effects of the commensal bacteria B. subtilis. However, it is not clear how specific cgt manipulations may affect total levels of glucosylceramides. Indeed, one study showed that only double-knockout mutants (cgt-3;cgt-1) demonstrated observable phenotypes (Marza et al. 2009); but, another showed pharmacological inhibition of glucosylceramides, particularly glucosylceramide transferase 2, actually increase lifespan (Cutler et al. 2014). Furthermore, the enzymes may have location-specific cell functions that have not been explored.
Previously, it was found that wild-type B. subtilis can promote tolerance to heavy metal, osmotic, oxidative, pathogenic, and temperature stress (Donato et al. 2017;Smolentseva et al. 2017). To examine the effect of mutations in cgt enzymes on adult stress response, we performed an oxidative stress assay by examining acute survival to the oxidative stressor paraquat (PQ) in 1, 5, and 10 day old animals. We found that wild-type N2 worms fed B. subtilis performed worse in response to 100mM PQ at 1-day old than N2 worms fed OP50 ( Figure 1C). This was also observed in cgt-1(tm1027) mutants. However, 5-day-old N2 worms fed B. subtilis improved acute survival to PQ compared to those who fed OP50 (p=0.007; Figure 1C); however, this effect was not observed at 10-days of age. The improved response to PQ in 5-day old wild-type animals was not observed in the presence of cgt mutations.
In summary, we show that loss of individual CGTs impact does not block the lifespan extending effects of the bacteria B. subtilis. Prior research demonstrates that single CGT knockouts may play a minor role in C. elegans response to stress (Marza et al. 2009). However, others have found that cgt-3 RNAi alone can reduce survival to the oxidative stressor TBHP (Wang et al. 2021). Thus, further experiments deleting or knocking down multiple CGTs may provide further insight into the roles of glucosylceramides in host-bacterial interactions. It was interesting that there were some differences between 1 day and 5 day responses of wild-type animals fed OP50 vs B. subtilis to PQ (from a slightly detrimental effect to a slightly beneficial effect), which may suggest that colonization of B. subtilis is needed to impact hosts. Although speculative, this finding supports the model that biofilm formation in B. subtilis is necessary to promote host lifespan and physiology (Donato et al. 2017;Smolentseva et al. 2017). Nevertheless, our data suggests that CGTs may play minor roles in the beneficial effects of the commensal bacteria B. subtilis, unlike findings from pathogenic bacteria. Future work examining how sphingolipid enzymes affect intestinal membranes may inform our understanding of how bacteria, including those that form biofilms, impact host physiology.

Methods
Strains. Wild-type N2 animals were obtained from the Caenorhabditis Genetics Center (CGC), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Mutants for cgt-1(tm1027), cgt-2(tm1192), cgt-3(tm504) were obtained from the Mitani lab at the National BioResource Project and cgt-1(ok1045) was obtained from the Caenorhabditis Genetics Center (CGC). Strains were not backcrossed into lab strains of N2. All tm strains are thought to eliminate CGT function or enzymatic activity (Marza et al. 2009). ok1045 is a large ~1800 deletion spanning 7 exons (Wormbase). All worms were maintained on Nematode Growth Medium (Stiernagle 2006) and maintained at 20℃ on respective bacteria.